Recently, I was trying to evaluate the anti-proliferation activity of a new synthesis compound.
I used the MTT assay to determine the effective concentration at first. Then, I used the effective concentration to perform a trypan blue assay to calculate the number of cells and observed the cell type.
However, the inhibition result of the trypan blue assay is not consistent with the result of the MTT assay.
My question is why did it happen? Is there any other problem I neglected?
I believe this observation might be explained by the different mechanisms underlying trypan blue and MTT assays. For example, one compound might affect the metabolic activity (measured by MTT) but not affect membrane integrity (trypan blue).
Hi Sin-Min Li
Have you tried running the MTT assay with known cell numbers (i.e. - 500, 1000, 2000, etc) and plotted a straight line equation? Then you should be able to reverse the equation to calculate an unknown number of cells. If this works as expected, you might have an issue with your trypan blue (which can be very subjective).
Using only one method is better as, as mentioned by Daniel Carneiro Moreira , you are trying to compare membrane integrity and metabolic activity (which would also be potentiated by actively proliferating cells more than dormant cells).
Hello Sin-Min Li
MTT is not the best choice for estimating the number of viable cells in vitro because of several limitations.
1.It lacks sensitivity. The sensitivity may be improved by optimizing the concentration of MTT and incubation time. But the incubation time is limited due to the toxic nature of MTT reagent.
2.There may be chemical interference that could increase the background absorbance and assay artifacts. Chemicals like reducing compounds could cause non-enzymatic reduction of MTT to formazan.
3.MTT reagent itself could exhibit cytotoxic effects and adding MTT to estimate cell viability may actually be damaging to cells during the experiment.
4.MTT reduction reflects cell metabolism and not cell number. So, cells growing rapidly as a monolayer will have a different rate of metabolism than those cells growing as a confluent monolayer or have become senescent.
I recommend that to evaluate the anti-proliferation activity of your newly synthesized compound you try the Sulforhodamine B (SRB) cytotoxicity assay which is a sensitive, reproducible and easy-to-use assay based on the ability of SRB to bind to protein components of cells that have been fixed to tissue culture plates. SRB is a bright-pink amino xanthene dye with two sulfonic groups that bind to basic amino acid residues under mild acidic conditions and dissociate under basic conditions.
As the binding of SRB is stoichiometric, the amount of dye extracted from stained cells is directly proportional to the cell mass. The fixed dye is solubilized and is measured photometrically at OD 540 nm with a reference filter of 690 nm. The OD values correlate with total protein content and therefore with cell number.
Best.
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